Immunocytochemical and radioautographic evidence for secretion and intracellular degradation of enamel proteins by ameloblasts during the maturation stage of amelogenesis in rat incisors

Abstract

In the continuously erupting rat incisor the ameloblasts progress through distinct stages associated with the secretion and maturation of enamel. We have examined the possibility that the so-called "postsecretory" ameloblasts of the maturation stage of amelogenesis remain biosynthetically active and are engaged in the synthesis, secretion, and degradation of enamel gene products. The ultrastructural distribution of antigenic sites for enamel proteins was studied within enamel organ cells during the early maturation stage of amelogenesis in rat incisors by using the protein A-gold immunocytochemical technique and rabbit polyclonal antibodies developed against mouse amelogenins. All regions of amelogenesis from late secretion through the first complete modulation from ruffle-ended to smooth-ended ameloblasts were examined. Specific immunolabelling was found within the rough endoplasmic reticulum, Golgi saccules, secretory granules, and lysosomes of ameloblasts throughout these regions. The heaviest intracellular immunolabelling was found within secretory granules and lysosomes (multivesicular type). Quantitative analyses showed that the Golgi saccules and the multivesicular lysosomes of modulating ameloblasts were generally less immunoreactive compared to similar organelles in ameloblasts secreting the inner enamel layer. Radioautographic studies confirmed that ameloblasts of the maturation stage incorporated 3H-leucine and 3H-methionine and secreted labelled proteins into the enamel layer. Grain counts indicated that ameloblasts from the first ruffle-ended band incorporated about two-fold less 3H-methionine and secreted about tenfold less labelled proteins into the enamel compared to ameloblasts secreting the inner enamel layer. The results of this study confirm that ameloblasts do not terminate biosynthesis and secretion of enamel proteins once the final layer has been deposited on the surface of the developing enamel. They continue to form and release new proteins during the maturation stage which intermix with older proteins laid down initially during the secretory stage of amelogenesis. Secretory activity for enamel proteins has been detected in ameloblasts up to at least the second ruffle-ended phase of maturation, at which point the enamel matrix is partially soluble in EDTA.

Expression of Alternatively Spliced RNA Transcripts of Amelogenin Gene Exons 8 and 9 and Its End Products in the Rat Incisor

“…A similar pattern of amelogenin mRNA expression has been reported to appear in rat molars (Fong et al 1996), hamster molars (Karg et al 1997), and mouse molars (Hu et al 2001). By electron microscopic immunocytochemistry of amelogenins combined with autoradiography, Nanci et al (1987) indicated that postsecretory maturation stage ameloblasts continue to synthesize and secrete minute amounts of amelogenins throughout the maturation stage, up to the point at which enamel matrix is lost by decalcification. In this experiment we could also localize intense immunoreactivity for 25-kD amelogenin in the supranuclear and distal cytoplasm of maturation stage ameloblasts (Figure 2d), indicating continued synthesis and secretion of enamel matrix proteins by the ameloblasts in early stages of enamel maturation.…”

Immunocytochemistry of matrix proteins in calcified tissues: functional biochemistry on section

“…Albumin-gold complexes have been used to study vascular permeability (Ghitescu et al 1988) and to visualize endocytotic activity in calcified tissue cells ). Finally, colloidal gold labeling can be combined with radioautography to provides a time dimension component to cytochemical detections (Figure 4 A) or with metal-based enzyme cytochemistry to identify the nature of subcellular compartments in which proteins are found (Figure 4 B) (Nanci et al 1987a).…”

Les protéines de l'émail dentaire - Etude immunocytochimique de l'amélogenèse-

“…Dans cet article, nous présentons une synthèse de nos travaux immunocytochimiques sur 1 'amé logenèse [12,13].…”

“…Un mar quage immunocytochimique spé cifique est détecté sur les amélo blastes au stade de sécrétion [ 12,13]. Des particules d'or apparais sent sur le réticulum endoplasmi que rugueux, les saccules de l'appareil de Golgi, les granules de sécrétion immatures et ma tu res associés au Golgi et les gra nules qui s'accumulent dans le prolongement de Tomes*** (figure 1).…”

“…La matrice extracellu laire de l'émail est fo rtement mar- • Amélo blastes de maturation. Au début de la phase de maturation, les deux types d'améloblastes, à surface lisse et invaginée, mon trent un marquage immunocyto chimique spécifique [13). Leurs lysosomes et corps multivésiculai res contiennent des particules d'or (fi�ure 2) .…”